Genes and genetic elements associated with sensitivity to chemotherapeutic drugs

ABSTRACT

The invention provides genetic suppressor elements that confer upon a cell resistance to one or more chemotherapeutic drug, methods for identifying and obtaining such elements, and methods of using such elements. The invention also provides cloned genes associated with sensitivity to chemotherapeutic drugs.

This invention was made with government support under grants CA39365 andCA-56738 by the National Institutes of Health. The government hascertain rights in the invention.

This application is a divisional of U.S. Ser. No. 08/480,552, filed Jun.7, 1995, now U.S. Pat. No. 5,665,550, issued Sep. 9, 1997, which is acontinuation of U.S. Ser. No. 08/033,086, filed Mar. 9, 1993, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 08/039,385[international application PCT/US91/07492, filed as internationalapplication on Oct. 11, 1991], which is a continuation-in-part of U.S.Ser. No. 07/599,730, filed Oct. 19, 1990, now U.S. Pat. No. 5,217,889,issued Jun. 8, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to genetic factors associated with sensitivity tochemotherapeutic drugs. More particularly, the invention relates tomethods for identifying such factors as well as to uses for suchfactors.

2. Summary of the Related Art

A broad variety of chemotherapeutic agents are used in the treatment ofhuman cancer. For example the textbook CANCER: Principles & Practice OfOncology, 2d Edition, (De Vita et al., Eds.), J. B. Lippincott company,Philadelphia, Pa. (1985) discloses as major antineoplastic agents theplant alkaloids vincristine, vinblastine, vindesine, and VM-26; theantibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin,mitomycin C and bleomycin; the antimetabolites methotrexate,5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine,cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylatingagents cyclophosphamide, melphalan, busufan, CCNU, MeCCNU, BCNU,streptozotocin, chlorambucil, cis-diamminedichloroplatinum,azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSAand mitoxantrone.

These and other chemotherapeutic agents such as etoposide and amsacrinehave proven to be very useful in the treatment of cancer. Unfortunately,some tumor cells become resistant to specific chemotherapeutic agents,in some instances even to multiple chemotherapeutic agents. Such drugresistance or multiple drug resistance can theoretically arise fromeither the presence of genetic factors that confer resistance to thedrugs, or from the absence of genetic factors that confer sensitivity tothe drugs. The former type of factors have been identified, and includethe multiple drug resistance gene mdr-1 (see Chen et al., Cell47:381-389 (1986)). However, the latter type of factor remains largelyunknown, perhaps in part because such absence of factors would tent tobe a recessive trait.

Identification of genes associated with sensitivity to chemotherapeuticagents is desirable, because the discovery of such genes can lead toboth diagnostic and therapeutic approaches for cancer cells and for drugresistant cancer cells, as well as to improvements in gene therapy andrational drug design. Recently, some developments have been made in thedifficult area of isolating recessive genetic elements, including oneinvolved in cytotoxic drug sensitivity. Roninson et al, U.S. Pat. No.5,217,889 (Ser. No. 07/599,730 issued Jun. 8, 1993) teaches generalizedmethod for obtaining genetic suppressor elements (GSEs), which aredominant negative factors that confer the recessive-type phenotype forthe gene to which the particular GSE corresponds. (See also Holzmayer etal., Nucleic Acids Res. 20:711-717 (1992)). Gudkov et al., Proc. Natl.Acad. Sci. USA 90:3231-3235 (1993) teaches isolation of GSEs inducingresistance to topoisomerase II-interactive drugs from topoisomerase IIcDNA. However, there remains a need for identifying yet unknown genes orgenetic elements associated with sensitivity to chemotherapeutic agents,a task made more difficult by the unavailability of a cloned gene asstarting material for preparing GSEs. Preferably, such genes or geneticelements will be involved in a common pathway that is implicated insensitivity to more than one chemotherapeutic agent. Most preferably,such genes or genetic elements will be identified by direct selection ofGSEs causing loss of the drug sensitivity phenotype.

BRIEF SUMMARY OF THE INVENTION

The invention provides genetic suppressor elements (GSEs) that conferupon cells resistance to chemotherapeutic drugs. These GSEs are randomfragments derived from genes associated with sensitivity tochemotherapeutic drugs, although the nature of such genes can be quitesurprising.

In a first aspect, the invention provides a method for identifying GSEsthat confer resistance to any chemotherapeutic drug for which resistanceis possible. This method utilizes chemotherapeutic drug selection ofcells that harbor clones from a random fragment expression libraryderived from total cDNA and subsequent rescue of library inserts fromdrug-resistance cells. In a second aspect, the invention provides amethod for identifying and cloning genes that are associated withsensitivity to chemotherapeutic drugs, including genes that have notbeen previously discovered. This method comprises the steps of screeninga full length cDNA library with a GSE that confers upon cells resistanceto chemotherapeutic (or an oligonucleotide or polynucleotideconstituting a portion of such a GSE) and determining the nucleotidesequence of the cDNA insert of any positive clones obtained. In a thirdaspect, the invention provides a method for obtaining GSEs havingoptimized suppressor activity for a gene associated with sensitivity toa chemotherapeutic drug. This method utilized chemotherapeutic drugselection of cells that harbor clones from a random fragment expressionlibrary derived from DNA of a gene associated with sensitivity to thesame chemotherapeutic drug, and subsequent rescue of the library insertsfrom drug resistant cells. In a fourth aspect, the invention providessynthetic peptides and oligonucleotides that confer upon cellsresistance to chemotherapeutic drugs. These synthetic peptides andoligonucleotides are designed based upon the sequence of adrug-resistance conferring GSEs according to the invention.

In a fifth aspect, the invention provides a diagnostic assay for tumorcells that are resistant to one or more chemotherapeutic drug due to theabsence of expression or underexpression of a particular gene. Thisdiagnostic assay comprises quantitating the level of expression of theparticular gene product by a particular tumor cell sample to be tested.In a sixth aspect, the invention provides dominant selectable markersthat are useful in gene co-transfer studies. These dominant selectablemarkers are drug resistance-conferring GSEs according to the inventionoperably linked to appropriate transcriptional control elements. In aseventh aspect, the invention provides in vivo-selectable markers thatare useful both for gene therapy and for enhanced chemotherapy forcancer. Such in vivo selectable markers are transferred into bloodprogenitor cells, which are then used to repopulate the patient's bloodexclusively with cells that contain a co-transferred therapeutic gene,or for chemotherapy, just the chemotherapeutic drug resistanceconferring GSE. In a eighth aspect, the invention provides a startingpoint for the rational design of pharmaceutical products that are usefulagainst tumor cells that are resistant to chemotherapeutic drugs. Byexamining the structure, function, localization and pattern ofexpression of genes associated with sensitivity to chemotherapeuticdrugs, strategies can be developed for creating pharmaceutical productsthat will overcome drug resistance in tumor cells in which such genesare either not expressed or underexpressed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1L show the nucleotide sequences of twelve GSEs thatconfer etoposide resistance upon cells and that were derived fromtopoisomerase II DNA, using a single gene random fragment expressionlibrary, as described in Example 1. The GSEs shown are FIG. 1A clone 2V[SEQ. ID. NO. 1], FIG. 1B clone Σ11 [SEQ. ID. NO. 2], FIG. 1C clone 6[SEQ. ID. NO. 3, FIG. 1D clone 5 [SEQ. ID. NO. 4], FIG.1E clone Σ28[SEQ. ID. NO. 5], FIG. 1F clone Σ2 [SEQ. ID. NO. 6], FIG. 1G clone Σ20[SEQ. ID. NO. 7], FIG. 1H clone 39 [SEQ. ID. NO. 8], FIG. 1I clone 12S[SEQ. ID. NO. 9], FIG. 1J clone Σ8 [SEQ. ID. NO. 10], FIG. 1K cloneΣVPs2 [SEQ. ID. NO. 11], and FIG. 1L clone ΣVM [SEQ. ID. NO. 12].

FIGS. 2A and 2B shows a scheme for construction of RFEL from NIH 3T3cDNA. FIG. 2A shows the overall construction scheme. FIG. 2B showsnormalization of the cDNA fragments. In FIG. 2B, t represents totalunfractionated cDNA, s and d represent the single-stranded anddouble-stranded fractions separated by hydroxyapatite, time pointsindicate the period of reannealing, and tubulin, c-myc, and c-fosindicate the probes used in Southern hybridization with the total,single-stranded and double-stranded fractions.

FIGS. 3A and 3B shows the structure of the LNCX vector and the adaptorused for cDNA cloning. The nucleotide sequences are shown for theATG-sense [SEQ. ID. NO. 13] and ATG-antisense [SEQ. ID. NO. 14] strandsof the adaptor.

FIG. 4 shows the overall scheme for selecting cell lines containingchemotherapeutic drug resistance-conferring GSEs and rescuing the GSEsfrom these cells.

FIGS. 5A and 5B shows etoposide resistance conferred by preselectedvirus (FIG. 5A) and PCR analysis of the selected and unselectedpopulations (FIG. 5B).

FIG. 6 shown a scheme for recloning individual PCR-amplified fragmentsfrom etoposide resistant selected cells into the LNCX vector, asdescribed in Example 4.

FIGS. 7A and 7B demonstrates resistance to 350 ng/ml etoposide,conferred upon the cells by the GSEs VPA (FIG. 7A) and VP9-11 (FIG. 7B).

FIGS. 8A and 8B shows resistance to various concentrations of etoposide,conferred upon the cells by the GSE anti-khcs under an IPTG-induciblepromoter (FIG. 8A) and the scheme for this selection (FIG. 8B).

FIG. 9 shown the nucleotide sequence of the GSE anti-khcs [SEQ. ID. NO.15].

FIG. 10 shows the nucleotide sequence of the GSE VPA [SEQ. ID. NO. 16].

FIG. 11 shown the nucleotide sequence of the GSE VP9-11 [SEQ. ID. NO.17].

FIGS. 12A through 12C shows the nucleotide sequence of the most of thecoding region of the mouse khcs cDNA [SEQ. ID. NO. 18].

FIGS. 13A through 13D show the dot matrix alignments of khcs proteinsequence deduced from the nucleotide sequence in FIG. 12 with kinesinheavy chain sequences from human (FIG. 13A), mouse (FIG. 13B), squid(FIG. 13C) or the portion of mouse khcs encoded by the anti-khcs GSE(FIG. 13D).

FIGS. 14A through 14F shows the plating efficiency in the presence ofvarious chemotherapeutic drugs of NIH 3T3 cells infected with the LNCXvector (indicated by crosses) or with the LNCX vector containing theanti-khcs GSE (indicated by dots).

FIG. 15 demonstrates increased immortalization of primary mouse embryofibroblasts by infection with the LNCX vector containing the anti-khcsGSE, relative to cells infected with the LNCX vector alone or uninfected(control) cells.

FIG. 16 shown cDNA-PCR quantitative analysis of expression of the humankhcs gene in various unselected and etoposide-selected human HeLa cells.Lanes a shows results for clone CX(O), lands a' for clone CX(200), lanesb for clone Σ11(O), lanes b' for clone Σ11 (1000), lanes c for clone6(O), lanes c' for clone 6(1000), lanes d for clone Σ20(O) and lanes d'for clone Σ20 (1000). The numbers in parentheses for each clone nameindicate the concentration of etoposide (ng/ml) present in the growthmedia. Bands indicative of khcs expression are shown along with bandsfor β-2 microglobulin expression as an internal control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to means for suppressing specific gene functionsthat are associated with sensitivity to chemotherapeutic drugs. Theinvention provides genetic suppressor elements (GSEs) that have suchsuppressive effect and thus confer resistance to chemotherapeutic drugs.The invention further provides methods for identifying such GSEs, aswell as methods for their use.

In a first aspect, the invention provides a method for identifying GSEsthat confer resistance to any chemotherapeutic drug for which resistanceis possible. The GSEs identified by this method will be homologous to agene that is associated with sensitivity to one or more chemotherapeuticdrug. For purposes of the invention, the term "homologous to a gene" hastwo different meanings, depending on whether the GSE acts through anantisense or antigene mechanism, or rather through a mechanism ofinterference at the protein level. In the former case, a GSE that is anantisense or antigene oligonucleotide or polynucleotide is homologous toa gene if it has a nucleotide sequence that hybridizes underphysiological conditions to the gene or its mRNA transcript by Hoogsteenor Watson-Crick base-pairing. In the latter case, a GSE that interferswith a protein molecule is homologous to the gene encoding that proteinmolecule if it has an amino acid sequence that is the same as thatencoded by a portion of the gene encoding the protein, or that would bethe same, but for conservative amino acid substitutions. In either case,as a practical matter, whether the GSE is homologous to a gene isdetermined by assessing whether the GSE is capable of inhibiting orreducing the function of the gene.

The method according to this aspect of the invention comprises the stepof screening a total cDNA or genomic DNA random fragment expressionlibrary phenotypically to identify clones that confer resistance to achemotherapeutic drug. Preferably, the library of random fragments oftotal cDNA or genomic DNA is cloned into a retroviral expression vector.In this preferred embodiment, retrovirus particles containing thelibrary are used to infect cells and the infected cells are tested fortheir ability to survive in a concentration of a chemotherapeutic drugthat kills uninfected cells. Preferably, the inserts in the library willrange in size from about 100 b.p. to about 700 b.p. and more preferably,from about 200 b.p. to about 500 b.p. Most preferably, the randomfragment library will be a normalized library containing roughly equalnumbers of clones corresponding to each gene expressed in the cell typefrom which it was made, without regard for the level of expression ofany gene. However, normalization of the library is unnecessary for theisolation of GSEs that are homologous to abundantly or moderatelyexpressed genes. Once a clonal population of cells that are resistant tothe chemotherapeutic drug has been isolated, the library clone encodingthe GSE is rescued from the cells. At this stage, the insert of theexpression library may be tested for its nucleotide sequence.Alternatively, the rescued library clone may be further tested for itsability to confer resistance to chemotherapeutic drugs in additionaltransfection or infection and selection assays, prior to nucleotidesequence determination. Determination of the nucleotide sequence, ofcourse, results in the identification of the GSE. This method is furtherillustrated in Examples 2-5.

In a second aspect, the invention provides a method for identifying andcloning genes that are associated with sensitivity to chemotherapeuticdrugs, including genes that have not been previously discovered. This isbecause GSEs, or portions thereof, can be used as probes to screen fulllength cDNA or genomic libraries to identify their gene of origin. Insome cases, genes that are associated with sensitivity tochemotherapeutic drugs will turn out to be quite surprising. Forexample, GSEs that abrogate etoposide sensitivity are of a particularlysurprising nature. The target for etoposide is topoisomerase II, a DNAunwinding enzyme. GSEs prepared from random fragments of topoisomeraseII DNA do confer resistance to etoposide. Accordingly, it would beexpected that most GSEs conferring etoposide resistance would be derivedfrom DNA encoding toposisomerase II. Surprisingly, this is not the caseat all. Of three etoposide resistance-conferring GSEs obtained, two werederived from previously unidentified DNA sequences. A third such GSE wasderived from the kinesin heavy chain gene. Prior to this discovery,there was no suspicion that kinesin was in any way implicated inetoposide sensitivity. These results suggest that the method accordingto this aspect of the invention will provide much new and surprisinginformation about the genetic basis for resistance to chemotherapeuticdrugs. In addition, a kinesin-derived GSE conferring resistance toetoposide caused cellular effects suggesting that kinesin may beinvolved in programmed cell death. If this is indeed the case, then themethod according to this aspect of the invention also provides valuableinformation about the genetic basis for senescence and cell death. Thismay have important implications for studying genes involved indevelopment, since GSEs used to identify genes associated withchemotherapeutic drug resistance or senescence can also be expressed astransgenes in embryos to determine the role of such genes indevelopment. The method according to this aspect of the invention andits use for studying genes identified thereby and their cellular effectsare further illustrated in Example 6-8.

In a third aspect, the invention provides a method for obtaining GSEshaving optimized suppressor activity for a gene associated withsensitivity to a chemotherapeutic drug. In the method according to thisaspect of the invention, an initial GSE is obtained by the methodaccording to the first aspect of the invention. Then, the gene fromwhich the GSE is derived is identified and cloned by the methodaccording to the second aspect of the invention. This gene is thenrandomly fragmented and cloned into an expression vector, preferably aretroviral vector, to obtain a random fragment expression libraryderived exclusively from the gene of interest. This library is thentransferred to and expressed in mammalian cells, which are selected inthe presence of the appropriate chemotherapeutic drug. As a practicalmatter, such a library will contain a much greater variety of GSEsderived from the gene of interest than will a random fragment libraryprepared from total cDNA. Consequently, the likelihood of obtainingoptimized GSEs, as determined by maximized chemotherapeutic drugresistance, from the single gene random fragment library is much higher.A single gene random fragment library approach is shown in greaterdetail in Example 1.

In a fourth aspect, the invention provides synthetic peptides andoligonucleotides that are capable of inhibiting the function of genesassociated with sensitivity to chemotherapeutic drugs. Syntheticpeptides according to the invention have amino acid sequences thatcorrespond to amino acid sequences encoded by GSEs according to theinvention. Synthetic oligonucleotides according to the invention havenucleotide sequences corresponding to the nucleotide sequences of GSEsaccording to the invention. Once a GSE is discovered and sequenced, andits orientation is determined, it is straightforward to prepare anoligonucleotide corresponding to the nucleotide sequence of the GSE (forantisense-oriented GSEs) or amino acid sequence encoded by the GSE (forsense-oriented GSEs). In certain embodiments, such synthetic peptides oroligonucleotides may have the complete sequence encoded by the GSE ormay have only part of the sequence present in the GSE, respectively. Incertain other embodiments, the peptide or oligonucleotide may have onlya portion of the GSE-encoded or GSE sequence. In such latterembodiments, undue experimentation is avoided by the observation thatmay independent GSE clones corresponding to a particular gene will havethe same 5' or 3' terminus, but generally not both. This suggests thatmany GSEs have one critical endpoint, from which a simple walkingexperiment will determine the minimum size of peptide or oligonucleotidenecessary to inhibit gene function. For peptides, functional domains assmall as 6-8 amino acids have been identified for imunoglobulin bindingregions. Thus, peptides or peptide mimetics having these or largerdimensions can be prepared as GSEs. For antisense oligonucleotides,inhibition of gene function can be mediated by oligonucleotides havingsufficient length to hybridize to their corresponding mRNA underphysiological conditions. Generally, oligonucleotides having about 12 ormore bases will fit this description. Preferably, such oligonucleotideswill have from about 12 to about 100 nucleotides. As used herein, theterm oligonucleotide includes modified oligonucleotides havingnuclease-resistant internucleotide linkages, such as phosphorothioate,methylphosphonate, phosphorodithioate, phosphoramidate, phosphotriester,sulfone, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate and bridged phosphorothioate internucleotide linkages. Thesynthesis of oligonucleotides containing these modified linkages is wellknown in the art. (See e.g., Uhlmann and Peyman, Chemical Reviews90:543-584 (1990); Schneider and Banner, Tetrahedron Letters 31:335(1990). The term oligonucleotides also includes oligonucleotides havingmodified bases or modified ribose or deoxyribose sugars.

In a fifth aspect, the invention provides a diagnostic assay for tumorcells that are resistant to one or more chemotherapeutic drug due toabsence of expression or underexpression of a particular gene. By usingthe methods according to the first and second aspects of the inventionsuch a gene will be identified and cloned. To determine whether absenceof expression or underexpression of such a gene is a naturallyoccurring, and thus medically significant basis for chemotherapeuticdrug resistance, human tumor cells can be treated with cytotoxicquantities of an appropriate chemotherapeutic drug to select forspontaneous drug resistant mutants. These mutants can then be assessedfor their level of expression of the particular gene of interest.Absence of expression or significantly reduced expression indicates anatural mechanism of chemotherapeutic drug resistance. Accordingly, suchreduced or absent expression can be the basis for a diagnostic assay fortumor cell resistance to the chemotherapeutic drug or drugs of interest.A first embodiment of a diagnostic assay according to this aspect of theinvention utilizes an oligonucleotide or oligonucleotides that is/arehomologous to the sequence of the gene for which expression is to bemeasured. In this embodiment, RNA is extracted from a tumor sample, andRNA specific for the gene of interest is quantitated by standard filterhybridization procedures, an RNase protection assay, or by quantitativecDNA-PCR. (See Noonan et al, Proc. Natl. Acad. Sci. USA 87:7160-7164(1990)). In a second embodiment of a diagnostic assay according to thisaspect of the invention, antibodies are raised against a syntheticpeptide having an amino acid sequence that is identical to a portion ofthe protein that is encoded by the gene of interest. These antibodiesare then used in a conventional quantitative immunoassay (e.g., RIA orimmunohistochemical assays) to determine the amount of the gene productof interest present in a sample of proteins extracted from the tumorcells to be tested, or on the surface or at locations within the tumorcells to be tested.

In a sixth aspect, the invention provides dominant selectable markersthat are useful in gene co-transfer studies. Since GSEs according to theinvention confer resistance to chemotherapeutic drugs, the presence of avector that expresses the GSE can readily be selected by growth of avector-transfected cell in a concentration of the appropriate cytotoxicdrug that would by cytotoxic in the absence of the GSE. GSEs accordingto the invention are particularly well suited as dominant selectablemarkers because their small size allows them to be easily incorporatedalong with a gene to be cotranfered even into viral vectors havinglimited packaging capacity.

In a seventh aspect, the invention provides in vivo-selectable markersthat are useful both in gene therapy and in enhancing the effectivenessof chemotherapy. For gene therapy, GSEs according to the invention canbe co-transferred on a vector into human CD34⁺ blood progenitor cellsfrom a patient along with a therapeutic gene that, when expressed, willalleviate a genetic disorder. The cells can be selected in vitro forresistance to an appropriate chemotherapeutic drug, thereby assuringsuccessful transfer of the GSE, and by implication, of the therapeuticgene as well. The progenitor cells containing the GSE and therapeuticgene can then be returned to the patient's circulation. Finally, thecells containing the GSE and therapeutic gene can be selected in vivo byadministration of the appropriate chemotherapeutic drug (to which theGSE confers resistance) in a concentration that is cytotoxic to normalblood cells. In this way, those cells having the GSE and therapeuticgene will repopulate the patient's blood.

For enhancement of chemotherapy, a GSE according to the invention can betransferred alone or with another gene on an expression vector intoCD34⁺ blood progenitor cells taken from a cancer patient. In vitroselection of the progenitor cells harboring the GSE is then carried out,using the appropriate chemotherapeutic drug. The selected cells are thenreturned to the patient's circulation and allowed time to beginrepopulating the blood. After an appropriate period, aggressivechemotherapy can be carried out, using much higher than ordinaryconcentrations of an appropriate chemotherapeutic drug (to which the GSEconfers resistance), since toxic side effects to the immune system willbe avoided due to GSE expression in those cells.

In either of these therapeutic contexts, it may be desirable to have theGSE expressed in the progenitor cells (and subsequently in the bloodcells), only when its expression is beneficial, i.e., during in vitroselection of cells harboring the GSE and again during in vivo selectionor chemotherapy. To accomplish this, an inducible promoter can be usedto express the GSE. Then, the appropriate inducing agent is added to thecells prior to and during in vitro selection and again prior to andduring in vivo selection or chemotherapy. As long as the inducing agentis not normally present in the human body, the GSE will not be expressedat any other time.

In an eighth aspect, the invention provides a starting point for therational design of pharmaceutical products that can counteractresistance by tumor cells to chemotherapeutic drugs. The proteinsequence encoded by genes from which the GSEs were derived can bededuced from the cDNA sequence, and the function of the correspondingproteins may be determined by searching for homology with known genes orby searching for known functional motives in the protein sequence. Ifthese assays do not indicate the protein function, it can be deducedthrough the phenotypic effects of the GSEs suppressing the gene. Sucheffects can be investigated at the cellular level, by analyzing variousgrowth-related, morphological, biochemical or antigenic changesassociated with GSE expression. The GSE effects at the organism levelcan also be studied by introducing the corresponding GSEs as transgenesin transgenic animals (e.g. mice) and analyzing developmentalabnormalities associated with GSE expression. The gene function can alsobe studied by expressing the full-length cDNA of the corresponding gene,rather than a GSE, from a strong promoter in cells or transgenicanimals, and studying the changes associated with overexpression of thegene.

Full-length or partial cDNA sequences can also be used to direct proteinsynthesis in a convenient prokaryotic or eukaryotic expression system,and the produced proteins can be used as immunogens to obtain polyclonalor monoclonal antibodies. These antibodies can be used to investigatethe protein localization and as specific inhibitors of the proteinfunction, as well as for diagnostic purposes. In particular, antibodiesraised against a synthetic peptide encoded by part of the complement ofthe sequence of the GSE anti-khcs, or the corresponding region of thehuman KHCS protein should be particularly useful, as should antibodiesraised against an amino acid sequence encoded by part of the VPA orVP9-11 (see FIGS. 9-11).

Understanding the biochemical function of a gene involved in drugsensitivity is likely to suggest pharmaceutical means to stimulate ormimic the function of such a gene and thus augment the cytotoxicresponse to anticancer drugs. For example, if the gene encodes an enzymeproducing a certain compound, such a compound can be synthesizedchemically and administered in combination with cytotoxic drugs. If apharmaceutical approach is not apparent from the protein function, onemay be able to upmodulate gene expression at the level of transcription.This can be done by cloning the promoter region of the correspondinggene and analyzing the promoter sequence for the presence of ciselements known to provide the response to specific biologicalstimulators.

The most straightforward way to increase the expression of a drugsensitivity gene, identified through the GSE approach, would be toinsert a full-length cDNA for such a gene into a retroviral vector. Sucha vector, in the form of a recombinant retrovirus, will be delivered totumor cells in vivo, and, upon integration, would sensitize such cellsto the effects of the corresponding chemotherapeutic drug. A similarstrategy for selective delivery of a drug-sensitivity gene into ratbrain tumors, followed by curative treatment with the appropriate drug,was reported by Culver et al., Science 256:1550-1552 (1992). Theselective delivery to tumor cells can be accomplished on the basis ofthe selectivity of retrovirus-mediated transduction for diving cells.Alternatively, the selectivity can be achieved by driving the expressionof the drug sensitivity gene from a tissue- or tumor-specific promoter,such as, for example, the promoter of the carcinoembryonic antigen gene.

The protein structure deduced from the cDNA sequence can also be usedfor computer-assisted drug design, to develop new drugs that affect thisprotein in the same manner as the known anticancer drugs. The purifiedprotein, produced in a convenient expression system, can also be used asthe critical component of in vitro biochemical screening systems for newcompounds with anticancer activity. Accordingly, mammalian cells thatexpress chemotherapeutic drug resistance-conferring GSEs according tothe invention are useful for screening compounds for the ability toovercome drug resistance.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.

EXAMPLE 1 Development of GSEs for Human Topoisomerase II

Topoisomerase II is a DNA unwinding enzyme that serves as a target formany anti-cancer drugs, including etoposide, doxorubicin and amsacrine.The enzyme normally acts by double-strand DNA cleavage, followed bystrand passage and religation of the breaks. Anti-cancer drugs causetrapping of the enzyme in complexes having double-strand breaks heldtogether by the enzyme, thereby leading to lethal damage in replicatingcells. Some cell lines that are resistant to anti-cancer drugs thatinteract with topoisomerase II have decrease expression of this enzyme.

Random fragment selection of GSEs requires transfer of the expressionlibrary into a very large number of recipient cells. Therefore, toprepare a random fragment library containing GSEs for topoisomerase II,the efficient retroviral vector system was chosen. Overlapping cDNAclones spanning the entire coding sequence for topoisomerase II weremixed and randomly fragmented into 250-350 bp fragments by DNase I inthe presence of Mn⁺⁺ ions and fragment termini were filled in with T4DNA polymerase and Klenow fragment of DNA polymerase I. After ligationwith a synthetic adaptor providing translation initiation andtermination codons, the fragment mixture was amplified by PCR, usingadaptor-derived primers. The amplified mixture was cloned into the LNCXretroviral vector which contains a neo gene. (see Miller and Rosman,Biotechniques 7:980-986 (1989)).

A random fragment library containing 20,000 independent clones wasobtained, and was used to transfect amphotropic and ecotropicvirus-packaging cell lines derived from NIH 3T3 cells, to effectping-pong replication-mediated amplification of the virus (e.g., seeBodine, et al. Proc. Nalt. Acad. Sci. USA 87:3738-3742 (1990)). Thisresulted in a random fragment expression library (RFEL), a set ofrecombinant retroviruses containing a representative mixture of insertsderived from topoisomerase II gene sequences.

The uniformity of sequence representation in RFEL was monitored asfollows. NIH 3T3 cells were infected with virus-containing supernatant,followed 24 hours later by PCR amplification of integrated proviralinsert sequences in the presence of [³² P] alpha-dNTP. An aliquot of thePCR-amplified mixture was subjected to gel electrophoresis to establishthe absence of predominant bands. Another aliquot was used as a probefor a Southern bolt to topoisomerase II cDNA digested with severalfrequently cutting restriction enzymes. A representative sequencemixture was obtained, as evidenced by the absence of a predominant bandin the first test, and uniform hybridization to all fragments in thesecond test.

RFEL was then used to infect HeLa cells, and the infectants wereselected with G418. Colonies of G418-resistant cells, having about 50-70cells each, were then exposed to etoposide at a concentration of 200μg/ml. Approximately 50 of 10,000 G418-resistant colonies were etoposideresistant, compared to a frequency of <10⁻⁴ when insertless retroviruseswere used as a control. Cell lines were isolated frometoposide-resistant colonies. Amphotropic and ecotropic packaging celllines producing RFEL were also selected from etoposide resistance. Virusfrom etoposide resistant packaging cell lines was used to infect HeLacells, which were then selected with G418. G418-resistant infectantswere challenged with three topoisomerase II-interactive anticancerdrugs: etoposide, teniposide and amsacrine. A high proportion ofinfected cells were resistant to all three drugs, thus demonstratingthat etoposide selection of mouse packaging cell lines has led to thegeneration of GSEs active in both human and mouse cells. Theseinfectants were also used to establish cell lines. RFEL-derived insertswere recovered from etoposide resistant cell lines by PCR and reclonedinto LNCX vector. The newly-derived clones were then individually testedfor the ability to confer resistance to etoposide upon transfection intoHeLa cells, to confirm the GSE activity of the corresponding inserts.

Sequence analysis of 26 different isolated clones revealed that 16 ofthem were inserted in antisense and 10 in sense orientation. Of the 12GSEs confirmed, 7 were sense and 5 antisense, as shown in Table I. Thesequences of the confirmed GSEs are shown in FIGS. 1A through 1L. Thesense-oriented inserts of the confirmed GSEs encode 37-99 amino acidlong topo II-derived peptides, initiating either from the ATG codonprovided by the adaptor, or from the internal ATG codon within the openreading frame of Topoisomerase II, located close to the 5' end of theinsert in an appropriate context for translation initiation. Four of theconfirmed antisense GSEs come from the 3' third of the cDNA and one fromthe 5' end of cDNA, including the translation start site. Of thesense-oriented GSEs, five are derived from the central portion of theprotein that includes the active site tyrosine-804 that covalently bindsto DNA and the "leucine zipper" region involved in dimerization ofTopoisomerase II. One GSE peptide is derived from the region near theN-terminus and another from the region near the C-terminus of theprotein; no known functional sites are associated with either segment.

                  TABLE I                                                         ______________________________________                                        Confirmed Topoisomerase II-Derived GSE                                                     Orientation  Position in                                                                            Position                                     Clones (Sense/Antisense) cDNA.sup.2 of peptide.sup.b                        ______________________________________                                        2V       Antisense    -18-145                                                   Σ11 Sense 393-605 134-201                                               6 Sense 2352-2532 808-844                                                     5 Sense 2511-2734 846-911                                                     Σ28 Sense 2603-2931 879-977                                             Σ2 Antisense 3150-3343                                                  Σ20 Antisense 3486-3692                                                 39 Antisense 3935-4127                                                        12S Sense 4102-4343 1368-1447                                                 ΣVPs2 Sense 2494-2834 846-944                                           Σ8 Antisense 4123-4342                                                  ΣVM Sense 2501-2670 846-890                                           ______________________________________                                         .sup.a Position in the cDNA sequence of topoisomerase II; residues            numbered as in TsaiPflugfelder et al., Proc. Natl. Acad. Sci. USA             85:7177-7181 (1988).                                                          .sup.b Position of the peptide encoded by senseoriented GSEs in the amino     acid sequence of topoisomerase II; translation assumed to initiate from       the first ATG codon in the correct open reading frame.                   

These results demonstrate that GSEs that act according to multiplemechanisms to confer etoposide resistance can be prepared from a randomfragment library of DNA encoding topoisomerase II. In addition, theseresults show that GSEs produced from one mammalian species can be activein another mammalian species.

EXAMPLE 2 Generation of a Normalized Random Fragment cDNA Library in aRetroviral Vector

As shown in FIGS. 2A and 2B a normalized cDNA population was preparedusing a modification of the protocol of Patanjali et al., Proc. Natl.Acad. Sci. USA 88:1943-1947 (1991). Poly(A)⁺ RNA was extracted from NIH3T3 cells. To obtain mRNAs for different genes expressed at variousstages of the cell growth, one half of the RNA was isolated from arapidly growing culture and the other half from quiescent cells that hadreached complete monolayer confluency. To avoid overrepresentation ofthe 5'-end sequences in a randomly primed cDNA population, RNA wasfragmented by boiling to an average size range 600-1,000 nucleotides.These RNA fragments were then used for preparing randomly primeddouble-stranded cDNA. This randomly primed cDNA was then ligated to asynthetic adaptor providing ATG codons in all three possible readingframes and in a proper context for translation initiation. The structureof the adaptor (see FIG. 3B) determined its ligation to the blunt-endedfragments of the cDNA in such a way that each fragment started frominitiation codons independently from its orientation. The adaptor wasnot supplied with termination codons in the opposite strand since thecloning vector pLNCX, contained such codons immediately downstream ofthe cloning site. This vector has been described by Miller and Rosman,Biotechniques 7:980-986 (1989). The ligated mixture was amplified byPCR, using the "sense" strand of the adaptor as a PCR primer, incontrast to the method of Patanjali et al., which utilized cloning theinitial cDNA preparation into a phage vector and then usingvector-derived sequences as PCR primers to amplify the cDNA population.The PCRs were carried out in 12 separate reactions that weresubsequently combined, to minimize random over- or under-amplificationof specific sequences and to increase the yield of the product. ThePCR-amplified mixture was size-fractionated by gel electrophoresis, and200-500 bp fragments were selected for subsequent manipulations incontrast to Patanjali's fragment size range of from 400 to 1,600 bp.

For normalization, the cDNA preparation was denatured and reannealed,using different time points, as described by Patanjali et al., supra,and shown in FIGS. 2A and 2B, for reannealing. The single-stranded anddouble-stranded DNAs from each reannealed mixture were separated byhydroxyapatite chromatography. The single-stranded DNA fractions fromeach time point of reannealing were PCR-amplified using theadaptor-derived primer and analyzed by Southern hybridization for therelative abundance of different mRNA sequences. The fraction thatcontained similar proportions of tubulin, c-myc and c-foc cDNA sequences(see FIGS. 2A and 2B), corresponding to high-, medium- and low-expressedgenes, respectively, was used for the library preparation.

The normalized cDNA preparation was cloned into the Cla I site of theMoMLV-based retroviral vector pLNCX, which carries the neo (G418resistance) gene, transcribed from the promoter contained in theretroviral long terminal repeat (LTR), and which expresses the insertedsequence from a strong promoter of the cytomegalovirus (CMV) (see FIG.3A). The ligation mixture, divided into five portions, was used for fivesubsequent large-scale transformations of E. coli. The transformedbacteria were plated on the total of 500 agar plates (150 mm indiameter) and the plasmid population (18 mg total) was isolated from thecolonies washed off the agar. A total of approximately 5×10⁷ clones wereobtained, more than 60% of which carried the inserts of normalized cDNA,as estimated by PCR amplification of inserts from 50 randomly pickedcolonies. These results demonstrate the feasibility of generating anormalized cDNA library of as many as 3×10⁷ recombinant clones in aretroviral plasmid expression vector.

EXAMPLE 3 Transduction of a Retroviral Random Fragment Library intoVirus-Packaging Cell Lines and NIH 3T3 Cells

The plasmid library prepared according to Example 2 was converted into amixture of retroviral particles by transfection into virus-packagingcells (derivatives of NIH 3T3) that express retroviral virion proteins.Examples of such cell lines have been described by Markowitz et al.,Virology 167:400-406 (1988). Ecotropic and amphotropic virus-packagingcell lines, GP+E86 and GP+envAm12, respectively, were mixed at a 1:1ratio, and 10⁷ cells of this mixture were transfected with the plasmidlibrary under standard calcium phosphate coprecipitation conditions.This transfection resulted in the packaging and secretion of ecotropicand amphotropic virus particles, which rapidly spread through thepackaging cell population, since ecotropic viruses are capable ofinfecting amphotropic packaging cells and vice versa. The yield of thevirus, as measured by the number of G418-resistant colonies obtainedafter the infection of NIH 3T3 cells, reached 10⁵ infectious units per 1ml of media during the stage of transient transfection (1-3days), thendecreased (4-8 days) and then rapidly increased due to the expression ofproviral genomes that became stably integrated in most of the packagingcells. The yield of the virus 9-12 days after transfection reached >10⁶per 1 ml of media supernatant. At this stage, the library showed fairlyeven representation of different fragments, but at later stagesindividual virus-producing clones began to predominate in thepopulation, leading to uneven representation of cDNA-derived inserts.The uniformity of sequence representation in the retroviral populationwas monitored by rapid extraction of DNA from cells infected with thevirus-containing supernatant, followed by PCR amplification of inserts.The inserts were analyzed first by the production of a continuous smearin ethidium-bromide stained agarose gel and then by Southernhybridization with different probes, including topiosomerase II, c-mycand tubulin. As long as each gene was represented by a smear of multiplefragments, the representativity of the library was considered to besatisfactory.

In other experiments, for transducing the random-fragment normalizedcDNA library into NIH 3T3 cells, without loss of representativity, NIH3T3 cells were infected either with a virus produced at the transientstage of transfection (days 1-3), or with the high-titer virus collected10-12 days after transfection. In the latter case, 100 ml of viralsuspension contained more than 10⁸ infectious units. In the case of the"transient" virus, NIH 3T3 cells were infected with at least 10⁷recombinant retroviruses by using 500 ml of media from virus-producingcells (five rounds of infection, 100 ml of media in each). These resultsdemonstrate the feasibility of converting a large and complex randomfragment library into retroviral form and delivering it to anon-packaging cell line without loss of complexity.

EXAMPLE 4 Isolation of GSEs Conferring Resistance to theChemotherapeutic Drug Etoposide

The overall scheme for the selection of GSEs conferring etoposideresistance is illustrated in FIG. 4. This selection was carried outdirectly on virus-producing packaging cells, in the expectation thatcells whose resistant phenotype is caused by the GSE expression willproduce virus particles carrying such a GSE. The mixture of amphotropicand ecotropic packaging cells was transfected with the cDNA library inthe LNCX vector, prepared according to Example 2 and the virus wasallowed to spread through the population for 9 days. Analysis of a smallpart of the population for G418 resistance showed that practically 100%of the cells carried the neo-containing provirus. The cells were thenexposed to 350 ng/ml etoposide for 15 days and then allowed to growwithout drug for two more weeks. No difference was observed between thenumbers of colonies obtained in the experiment and in the control(uninfected cells or cells infected with the insert-free LNCX virus)after etoposide selection. The virus present in the media supernatant ofthe surviving cells was then used to infect NIH 3T3 cells followed byetoposide selection using essentially the same protocol. NIH 3T3 cellsinfected with the library-derived virus produced by packaging cells thatwere selected with etoposide showed a major increase in the number ofetoposide-resistant cells relative to the control cells infected withthe insert-free LNCX virus, indicating the presence of biologicallyactive GSEs in the preselected virus population (see FIG. 5A).

The proviral inserts contained in the etoposide-selected NIH 3T3 cellswere analyzed by PCR. This analysis (see FIG. 5B) showed an enrichmentfor specific fragments, relative to the unselected population of theinfected cells. Individual PCR-amplified fragments were recloned intothe LNCX vector in the same position and orientation as in the originalplasmid, as illustrated in FIG. 6. A total of 42 proviral inserts,enriched after etoposide selection, were thus recloned, and testedeither in batches or individually for the ability to confer increasedetoposide resistance after retroviral transduction into NIH 3T3 cells.Three non-identical clones were found to induce etoposide resistance,indicating that they contained biologically active GSEs. Etoposideresistance induced by these clones is illustrated in FIGS. 7A and 7B andFIGS. 8A and 8B. These GSEs were named anti-khcs, VPA and VP9-11.

The ability of one of these GSEs (anti-khcs) to induce etoposideresistance was further documented by using the isopropylβ-D-thiogalactopyranoside (IPTG)-inducible promoter/activator system, asdescribed by Baim et al., Proc. Natl. Acad. Sci. USA 88:5072-5076(1991). The components of this system include an enhancer-dependentpromoter, combined in cis with multiple repeats of the bacterial lacoperator, and a gene expressing LAP267, an artificial regulatory proteinderived from the lac repressor and a mammalian transcriptionalactivator. The anti-khcs GSE was cloned into the plasmid pX6.CLN, whichcontains the inducible promotor used by Baim et al., supra. (a gift ofDr. T. Shenk) which expresses the inserts from an enhancerless SV40early gene promoter supplemented with 21 repeats of the lac operatorsequence. The resulting plasmid, which contains no selectable markers,was co-transfected into NIH 3T3 cells together with the LNCX plasmidcarrying the neo gene. The mass population of G418-selectedtransfectants, along with control cells transfected with the insert-freevector, was exposed to increasing concentrations of etoposide, in thepresence or in the absence of 5 mM IPTG. Even though the co-transfectionprotocol usually leads to the integration of the GSE in only a fractionof the G418-resistant cells, transfection with anti-khcs resulted in aclearly increased etoposide resistance, which was dependent on IPTG (seeFIGS. 8A and 8B).

EXAMPLE 5 Sequence Analysis of GSEs Conferring Resistance to theChemotherapeutic Drug Etoposide

The GSEs anti-khcs, VPA, and VP9-11, cloned as described in Example 4,were sequenced by the standard dideoxy sequencing procedure, and thededuced sequences are shown in FIGS. 9-11. The nucleotide sequences ofthe "sense" and "antisense" strands, as well as amino acid sequences ofthe peptides encoded by these strands, were analyzed for homology to thenucleic acid and protein sequences present in the National Center forBiotechnology Information data base, using the BLAST network program forhomology search. No significant homology with any sequence was detectedfor the GSEs VPA and VP9-11. IN contrast, the sequence corresponding tothe "antisense" strand of the anti-khcs GSE, showed strong homology withseveral genes encoding the heavy chain of kinesins, a family ofmicrotubule motor proteins involved in intracellular movement oforganelles or macromolecules along the microtubules of eukaryotic cells.This protein family has been reviewed by Endow, Trends Biochem. Sci.16:221-225 (1991). The highest homology was found with the human kinesinheavy chain (KHC) gene, as described by Navone et al., J. Cell Biol.177:1263-1275 (1992). Anti-khcs therefore encodes antisense RNA for amouse khc gene, which we term khcs for khc associated with sensitivity(to drugs) or senesence. We refer to the kinesin molecule, formed by theassociated of the Khcs protein with kinesin light chains, as kinesin-S,to distinguish it from the other kinesins present in mammalian cells.These results demonstrate that chemotherapeutic drug selection for GSEscan lead to the discovery of novel genetic elements, and can also revealroles of genes in drug sensitivity that had never before been suspected.

EXAMPLE 6 Cloning and Analysis of the Gene from which Anti-khcs GSE Genewas Derived

The anti-khcs GSE isolated in Example 4 was used as a probe to screen400,000 clones from each of two cDNA libraries in the lambda gt10vector. These libraries were prepared by conventional procedures fromthe RNA of mouse BALB/c 3T3 cells, either unsychronized or at G0→G1transistion, as described by Lan and Nathans, EMBO J. 4:3145-3531 (1985)and Proc. Natl. Acad. Sci. USA 84:1182-1186 (1987) (a gift of Dr. L.Lau). Screening of the first library yielded no hybridizing clones, buttwo different clones from the second library were found to containanti-khcs sequences. These clones were purified and sequenced. Sequenceanalysis showed that we have isolated the bulk of the mouse khcs cDNA,corresponding to 796 codons (the full-length human KHC cDNA encodes 963amino acids). This sequence is shown in FIGS. 12A through 12C. Themissing 5' and 3' terminal sequences are currently being isolated usingthe "anchored PCR" technique, as described by Ohara et al., Proc. Natl.Acad. Sci. USA 86:5673-5677 (1989).

The dot-matrix alignment of the sequenced portion of the khcs proteinwith previously cloned KHC proteins from the human (see Navone et al.,J. Cell. Biol. 117:1263-1275 (1992)), mouse (see Kato, J. Neurosci.2:704-711 (1991)) and squid (see Kosik et al., J. Biol. Chem.265:3278-3283 (1990)) is shown in FIGS. 13A through 13D. The portioncorresponding to the anti-khcs GSE, is shown in FIG. 9. The khcs gene ismost highly homologous to the human gene (97% amino acid identity),suggesting that the human KHC (KHCS) gene is functionally equivalent tothe mouse khcs. The alignment also shows that the anti-khcs GSEcorresponds to the region which is the most highly diverged betweendifferent kinesins. This result suggests that the anti-khcs GSE is anantisense-oriented GSE fragment with an inhibitory effect specific tokinesin-S and not to the other mouse kinesins. This suggests thepossibility that suppression of the other members of the kinesin familywould have a detrimental effect on cell growth, thus resulting inselective isolation of the most specific sequence within the khcs gene.

EXAMPLE 7 Assessment of Drug Cross-Resistance Conferred by Resistance toEtoposide

To determine the spectrum of drugs to which the anti-khcs GSE wouldconfer resistance, we have developed a stable population of ecotropicpackaging cells producing the LNCX virus with the anti-khcs insert. Thisvirus was used to infect NIH 3T3 cells. Two days after infection, thecells were analyzed for resistance to several different drugs, relativeto control cells infected with the LNCX vector virus, using the standardplating efficiency assay. FIGS. 14A through 14F shows one out of threesets of experiments carried out with different drugs by this assay.Cells infected with the anti-khcs virus showed a clear increase in theirresistance to etoposide and Adriamycin relative to control NIH 3T3 cellsinfected with the control LNCX virus. No changes in resistance wereobserved with colchicine, cisplatin, camptothecin, or actinomycin D.These latter results remain preliminary, however, because this assay,analyzing total unselected virus-infected populations in relativelyinsensitive, compared with analysis of highly expressingindividually-selected clones. Overall, these results demonstrate thatselection of GSEs that confer resistance to one chemotherapeutic drugcan result in obtaining GSEs that confer resistance to additionalchemotherapeutic drugs.

EXAMPLE 8 Assessment of Cellular Effects of GSEs that Confer Resistanceto Etoposide

The virus carrying the anti-khcs GSE was tested for the ability toincrease the life span of primary mouse embryo fibroblasts (MEF). MEFwere prepared from 10 day old mouse embryos by a standard trypsinizationprocedure and senescent cells were frozen at different passages prior tocrisis. Senescent MEF, two weeks before crisis, were infected withrecombinant retroviruses carrying LNCX vector either without an insertor with anti-khcs. FIG. 15 shows MEF cell colonies two weeks aftercrisis. Relative to uninfected MEF cells, or cells infected with acontrol LNCX virus, cells infected with anti-khcs showed a greatincrease in the proportion of cells surviving the crisis. Post-crisiscells infected with the anti-khcs virus showed no microscopticallyvisible features of neoplastic transformation. These results indicatethat anti-khcs promotes the immortalization of normal senescentfibroblasts. These results suggest that the normal function of kinesin-Smay be associated with the induction of programmed cell death occurringafter exposure to certain cytotoxic drugs or in the course of cellularsenescence. These results also indicate that isolation of GSEs thatconfer resistance to chemotherapeutic drugs can provide insight into thecellular genes and processes involved in cell growth regulation.

EXAMPLE 9 Assessment of the Role of Decreased khcs Gene Expression inNaturally Occurring Mechanisms of Drug Resistance

To test whether decreased khcs gene expression is associated with anynaturally occurring mechanisms of drug resistance, an assay wasdeveloped for measuring khcs mRNA levels by cDNA-PCR. This assay is amodification of the quantitative assay described by Noonan et al., Proc.Natl. Acad. Sci. USA 87:7160-7164 (1990) for determining mdr-1 geneexpression. The oligonucleotide primers had the sequencesAGTGGCTGGAAAACGAGCTA [SEQ. ID. No. 19] and CTTGATCCCTTCTGGTTGAT [SEQ.ID. No. 20]. These primers were used to amplify a 327 bp segment ofmouse khcs cDNA, corresponding to the anti-khcs GSE. These primersefficiently amplified the mouse cDNA template but not the genomic DNA,indicating that they spanned at least one intron in the genomic DNA.Using these primers, we determined that khcs mRNA is expressed at ahigher level in the mouse muscle tissue than in the kidney, liver orspleen.

In another experiment a pair of primers amplifying a homologous segmentof the human KHCS cDNA was selected, based on the reported human KHCsequence published by Navone et al., J. Cell. Biol. 117:1263-1275(1992). The sequences of these primers are AGTGGCTTGAAAATGAGCTC [SEQ.ID. No. 21] and CTTGATCCCTTCTGGTAGATC [SEQ. ID. No. 22], and theyamplify a 327 bp cDNA fragment. These primers were used to test forchanges in the KHCS gene expression in several independently isolatedpopulations of human HeLa cells, each selected for spontaneouslyacquired etoposide resistance. β₂ -microglobulin cDNA sequences wereamplified as an internal control. FIG. 16 shows the results of thecDNA-PCR assay on the following population: CX(0), HeLa populationinfected with the LNCX vector virus and selected with G418; CX (200),the same cells selected for resistance to 200 ng/ml etoposide; Σ11(0),6(0) and Σ21(0), populations obtained after infection of HeLa cells withrecombinant retroviruses carrying different GSEs derived fromtopoisomerase αcDNA, as described in Example 1, and selected with G418:Σ11(1000), 6(1000) and Σ21(1000), the same populations selected forresistance to 1 μg/ml etoposide. As shown in FIG. 16, the yield of thePCR product specific for the khcs gene was significantly lower in eachof the etoposide-selected populations than in the control cells. Thisresult indicates that a decrease in the khcs gene expression is a commonnatural mechanism for drug resistance.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 164 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GTGTCTGGGC GGAGCAAAAT ATGTTCCAAT TGTGTTTTCT TTTGATAGAT TC -            #TTTCAACA     60                                                                 - - GACAGTCTTT TCTTAGCATC TTCATTTTTC TTTATTTTGT TGACTTGCAT AT -            #TTTCATTT    120                                                                 - - ACAGGCTGCA ATGGTGACAC TTCCATGGTG ACGGTCGTGA AGGG   - #                      - #164                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 213 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TGAAAAGATG TATGTCCCAG CTCTCATATT TGGACAGCTC CTAACTTCTA GT -             #AACTATGA     60                                                                 - - TGATGATGAA AAGAAAGTGA CAGGTGGTCG AAATGGCTAT GGAGCCAAAT TG -            #TGTAACAT    120                                                                 - - ATTCAGTACC AAATTTACTG TGGAAACAGC CAGTAGAGAA TACAAGAAAA TG -            #TTCAAACA    180                                                                 - - GACATGGATG GATAATATGG GAAGAGCTGG TGA       - #                  -      #        213                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 181 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GCCCATTGGT CAGTTTGGTA CCAGGCTACA TGGTGGCAAG GATTCTGCTA GT -            #CCACGATA     60                                                                 - - CATCTTTACA ATGCTCAGCT CTTTGGCTCG ATTGTTATTT CCACCAAAAC AT -            #GATCACAC    120                                                                 - - GTTGAAGTTT TTATATGATG ACAACCAGCG TGTTGAGCCT GAATGGTACA TT -            #CCTATTAT    180                                                                 - - T                  - #                  - #                  - #                  181                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 224 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGAATGGTAC ATTCCTATTA TTCCCATGGT GCTGATAAAT GGTGCTGAAG GA -             #ATCGGTAC     60                                                                 - - TGGGTGGTCC TGCAAAATCC CCAACTTTGA TGTGCGTGAA ATTGTAAATA AC -            #ATCAGGCG    120                                                                 - - TTTGATGGAT GGAGAAGAAC CTTTGCCAAT GCTTCCAAGT TACAAGAACT TC -            #AAGGGTAC    180                                                                 - - TATTGAAGAA CTGGCTCCAA ATCAATATGT GATTAGTGGT GAAG   - #                      - #224                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 329 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TGCGTGAAAT TGTAAATAAC ATCAGGCGTT TGATGGATGG AGAAGAACCT TT -             #GCCAATGC     60                                                                 - - TTCCAAGTTA CAAGAACTTC AAGGGTACTA TTGAAGAACT GGCTCCAAAT CA -            #ATATGTGA    120                                                                 - - TTAGTGGTGA AGTAGCTATT CTTAATTCTA CAACCATTGA AATCTCAGAG CT -            #TCCCGTCA    180                                                                 - - GAACATGGAC CCAGACATAC AAAGAACAAG TTCTAGAACC CATGTTGAAT GG -            #CACCGAGA    240                                                                 - - AGACACCTCC TCTCATAACA GACTATAGGG AATACCATAC AGATACCACT GT -            #GAAATTTG    300                                                                 - - TTGTGAAGAT GACTGAAGAA AAACTGGCA         - #                  - #               329                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CACTCTTTTC AGTTTCCTTT TCGTTGTCAC TCTCTTCATT TTCTTCTTCA TC -             #TGGAACCT     60                                                                 - - TTTGCTGGGC TTCTTTCCAG GCCTTCACAG GATCCGAATC ATATCCCCTC TG -            #AATCAGAA    120                                                                 - - CTTTAATTAA TTCTTTCTTA GGCTTATTTT CAATGATTAT TTTGCCATCT AT -            #TTTCTCAT    180                                                                 - - AGATAAAGCG AGCC              - #                  - #                      - #    194                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 206 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TCTGCCTCTG CTTTCATTTC TATGGTTATT CGTGGAATGA CTCTTTGACC AC -             #GCGGAGAA     60                                                                 - - GGCAAAACTT CAGCCATTTG TGTTTTTTTC CCCTTGGCCT TCCCCCCTTT CC -            #CAGGAAGT    120                                                                 - - CCGACTTGTT CATCTTGTTT TTCCTTGGCT TCAACAGCCT CCAATTCTTC AA -            #TAAATGTA    180                                                                 - - GCCAAGTCTT CTTTCCACAA ATCTGA          - #                  - #                 206                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GACACGACAC TTTTCTGTGG TTTCAGTTCT TTGTTACTAA GTTTTGGGGA AG -             #TTTTGGTC     60                                                                 - - TTAGGTGGAC TAGCATCTGA TGGGACAAAA TCTTCATCAT CAGTTTTTTC AT -            #CAAAATCT    120                                                                 - - GAGAAATCTT CATCTGAATC CAAATCCATT GTGAATTTTG TTTTTGTTGC TG -            #CTCTCCGT    180                                                                 - - GGCTCTGTTT CTCG              - #                  - #                      - #    194                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 242 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CTGAAACCAC AGAAAAGTGT CGTGTCAGAC CTTGAAGCTG ATGATGTTAA GG -             #GCAGTGTA     60                                                                 - - CCACTGTCTT CAAGCCCTCC TGCTACACAT TTCCCAGATG AAACTGAAAT TA -            #CAAACCCA    120                                                                 - - GTTCCTAAAA AGAATGTGAC AGTGAAGAAG ACAGCAGCAA AAAGTCAGTC TT -            #CCACCTCC    180                                                                 - - ACTACCGGTG CCAAAAAAAG GGCTGCCCCA AAAGGAACTA AAAGGGATCC AG -            #CTTTGAAT    240                                                                 - - TC                  - #                  - #                  - #                 242                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 341 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AACCAGCGTG TTGAGCCTGA ATGGTACATT CCTATTATTC CCATGGTGCT GA -             #TAAATGGT     60                                                                 - - GCTGAAGGAA TCGGTACTGG GTGGTCCTGC AAAATCCCCA ACTTTGATGT GC -            #GTGAAATT    120                                                                 - - GTAAATAACA TCAGGCGTTT GATGGATGGA GAAGAACCTT TGCCAATGCT TC -            #CAAGTTAC    180                                                                 - - AAGAACTTCA AGGGTACTAT TGAAGAACTG GCTCCAAATC AATATGTGAT TA -            #GTGGTGAA    240                                                                 - - GTAGCTATTC TTAATTCTAC AACCATTGAA ATCTCAGAGC TTCCCGTCAG AA -            #CATGGACC    300                                                                 - - CAGACATACA AAGAACAAGT TCTAGAACCC ATGTTGAATG G    - #                      - #  341                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 220 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AATTCAAAGC TGGATCCCTT TTAGTTCCTT TTGGGGCAGC CCTTTTTTTG GC -             #ACCGGTAG     60                                                                 - - TGGAGGTGGA AGACTGACTT TTTGCTGCTG TCTTCTTCAC TGTCACATTC TT -            #TTTAGGAA    120                                                                 - - CTGGGTTTGT AATTTCAGTT TCATCTGGGA AATGTGTAGC AGGAGGGCTT GA -            #AGACAGTG    180                                                                 - - GTACACTGCC CTTAACATCA TCAGCTTCAA GGTCTGACAC     - #                      - #   220                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 170 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GTGTTGAGCC TGAATGGTAC ATTCCTATTA TTCCCATGGT GCTGATAAAT GG -             #TGCTGAAG     60                                                                 - - GAATCGGTAC TGGGTGGTCC TGCAAAATCC CCAACTTTGA TGTGCGTGAA AT -            #TGTAAATA    120                                                                 - - ACATCAGGCG TTTGATGGAT GGAGAAGAAC CTTTGCCAAT GCTTCCAAGT  - #                 170                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - AATCATCGAT GGATGGATGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CCATCCATCCATCGATGATTAAA             - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 327 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - CTTGATCCCT TCTGGTTGAT GCCAGAAGCT CTTCCTGATC CAGCATTTGT AT -             #CTTCAATT     60                                                                 - - TCTCTACCAA TTGGCTTTGT TGGTTAATCT CTTCATCCTT GTCATCAAGT TG -            #TTTATACA    120                                                                 - - ATTTAGCAAG TTCTTCTTCA CACTTTCTTC TTTCAGCATC GGTAAAACTA CC -            #AGCCATTC    180                                                                 - - CGACTGCAGC AGCTGGTTTA TCACTGGTAA TAGCAATATC TTTATCCGCT GT -            #GAAGGCTT    240                                                                 - - CCAAATTAGC TTTCTCTTTG TCAAACTGCT CATCAATAGG CACTGTCTCC CC -            #GTTACGCC    300                                                                 - - AACGGTTTAG CTCGTTTTCC AGCCACT          - #                  - #                327                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 250 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CCGACCGGGA GCGGGAGAAG GAGCGGGAGC GGGAGCAGCG GGAGAAGGAG CG -             #GGAGAAGG     60                                                                 - - AGCTGGAGCG CGACGGGAGA AGGAACGGGA GCGCGAGCTG GAGCGGCAGC GG -            #GAGCAGCG    120                                                                 - - GGCGAGGGAG AAGGAGCTGC TGGCTGCCAA GGCCTTAGAG CCCACCACCT TC -            #CTGCCTGT    180                                                                 - - GGCCGAGCTG CACGGACTCC GAGGTCACAG CACGGAGGAG CGGCCCAAGC CC -            #TCGGAGCA    240                                                                 - - GCTGACCCCA                - #                  - #                      - #       250                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CTCAGAGGTG ATCCTCTCGG AGTCGAGCTC AGGAGAAGGA GTCCCCTTCT TT -             #GAGACTTG     60                                                                 - - GATGCAGACC TGCATGTCCG AGGAGGGCAA GATTTTGAAC CCTGACCATC CC -            #TGCTTCCG    120                                                                 - - CCCTGACTCC ACCGAAGTCG AGTCCTTGGT GGCCCTGCTC AACAACTCTT CA -            #GAGATGAA    180                                                                 - - GCTAGTACAG ATGAAGTAGC ACGAGGCC         - #                  - #                208                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2389 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - CGACAAACAT CATCTGGGAA GACCCACACG ATGGAGGGTA AACTTCATGA TC -             #CAGAAGGC     60                                                                 - - ATGGGAATTA TTCCAAGAAT AGTGCAAGAT ATTTTTAATT ATATTTACTC CA -            #TGGATGAA    120                                                                 - - AATTTGGAAT TTCATATTAA GGTTTCATAT TTTGAAATAT ATTTGGATAA GA -            #TAAGGGAC    180                                                                 - - TTGTTAGATG TTTCAAAGAC TAACCTTTCA GTCCATGAAG ACAAAAACCG TG -            #TTCCCTAT    240                                                                 - - GTAAAGGGGT GCACAGAACG TTTCGTGTGT AGTCCAGATG AAGTCATGGA TA -            #CCATAGAT    300                                                                 - - GAAGGGAAAT CCAACAGAGA TGTCGCAGTT ACAAATATGA ATGAACATAG CT -            #CTAGGAGC    360                                                                 - - CACAGCATAT TTCTTATTAA TGTAAAACAA GAGAATACAC AAACGGAACA GA -            #AACTCAGT    420                                                                 - - GGAAAGCTTT ATCTGGTTGA TTTAGCTGGC AGTGAGAAGG TTAGTAAGAC TG -            #GGGCTGAA    480                                                                 - - GGTGCTGTGC TGGATGAAGC TAAGAACATC AAGAAGTCAC TTTCTGCACT TG -            #GAAATGTC    540                                                                 - - ATTTCTGCTT TGGCAGAGGG CAGTACCTAT GTTCCTTATC GAGATAGTAA AA -            #TGACCAGA    600                                                                 - - ATTCTTCAAG ATTCATTAGG TGGCAACTGT AGGACCACTA TTGTCATATG CT -            #GCTCTCCA    660                                                                 - - TCATCATACA ATGAGTCTGA GACAAAGTCA ACACTCCTCT TTGGTCAAAG GG -            #CCAAAACA    720                                                                 - - ATTAAGAACA CAGTCTGTGT CAATGTAGAG TTAACTGCAG AGCAGTGGAA AA -            #AGAAGTAT    780                                                                 - - GAAAAAGAAA AGGAAAAAAA TAAGACTCTA CGGAACACTA TTCAGTGGCT GG -            #AAAACGAG    840                                                                 - - CTAAACCGTT GGCGTAACGG GGAGACAGTG CCTATTGATG AGCAGTTTGA CA -            #AAGAGAAA    900                                                                 - - GCTAATTTGG AAGCCTTCAC AGCGGATAAA GATACTGCTA TTACCAGTGA TA -            #AACCAGCT    960                                                                 - - GCTGCAGTCG GAATGGCTGG TAGTTTTACC GATGCTGAAA GAAGAAAGTG TG -            #AAGAAGAA   1020                                                                 - - CTTGCTAAAT TGTATAAACA GCTTGATGAC AAGGATGAAG AGATTAACCA AC -            #AAAGCCAA   1080                                                                 - - TTGGTAGAGA AATTGAAGAC ACAAATGCTG GATCAGGAAG AGCTTCTGGC AT -            #CAACCAGA   1140                                                                 - - AGGGATCAAG ATAATATGCA AGCTGAACTG AATCGCCTCC AAGCAGAAAA TG -            #ATGCTTCT   1200                                                                 - - AAAGAAGAAG TCAAAGAAGT TTTACAGGCC TTAGAGGAAC TGGCTGTTAA TT -            #ATGATCAG   1260                                                                 - - AAGTCTCAGG AAGTTGAAGA CAAAACAAAG GAATATGAAT TGCTTAGTGA TG -            #AATTGAAT   1320                                                                 - - CAAAAATCTG CAACTTTAGC AAGTATTGAT GCTGAGCTTC AGAAGCTGAA GG -            #AAATGACC   1380                                                                 - - AACCACCAGA AGAAACGAGC AGCTGAAATG ATGGCATCAT TATTAAAAGA CC -            #TTGCAGAA   1440                                                                 - - ATAGGAATTG CTGTGGGGAA TAACGATGTG AAGCAACCAG AAGGAACTGG TA -            #TGATAGAT   1500                                                                 - - GAAGAGTTTA CTGTTGCAAG ACTCTACATT AGCAAAATGA AATCAGAAGT AA -            #AGACCATG   1560                                                                 - - GTGAAACGCT GCAAACAGCT AGAAAGCACG CAGACTGAGA GCAACAAAAA AA -            #TGGAAGAA   1620                                                                 - - AATGAGAAAG AGTTAGCAGC ATGCCAGCTT CGGATCTCCC AACATGAAGC CA -            #AAATCAAG   1680                                                                 - - TCACTGACTG AGTACCTTCA GAATGTAGAA CAAAAGAAGA GGCAGCTGGA GG -            #AATCTGTT   1740                                                                 - - GATTCCCTTG GTGAGGAGCT AGTCCAACTC CGAGCACAAG AGAAAGTCCA TG -            #AAATGGAA   1800                                                                 - - AAAGAGCACT TGAACAAGGT TCAGACTGCA AATGAAGTCA AGCAAGCTGT TG -            #AGCAGCAG   1860                                                                 - - ATCCAGAGTC ACAGAGAAAC CCACCAAAAA CAAATCAGTA GCTTGCGAGA TG -            #AAGTTGAG   1920                                                                 - - GCAAAGGAAA AGCTAATCAC TGACCTCCAA GACCAAAACC AGAAGATGGT GT -            #TGGAGCAG   1980                                                                 - - GAACGGCTAA GGGTGGAGCA TGAGAGGCTG AAGGCTACAG ACCAAGAGAA GA -            #GCAGGAAG   2040                                                                 - - CTGCATGAGC TCACGGTTAT GCAAGACAGA CGAGAACAAG CAAGACAAGA CT -            #TGAAGGGT   2100                                                                 - - TTGGAGGAGA CCGTGGCAAA AGAACTTCAG ACTTTACACA ACCTGCGTAA GC -            #TCTTTGTT   2160                                                                 - - CAGGACTTGG CTACCAGGGT GAAAAAGAGG CCGAGGTCGA CTCTGACGAC AC -            #TGGCGGCA   2220                                                                 - - GTGCTGCACA GAAGCAGAAA ATCTCCTTCC TTGAAAACAA CCTTGAACAG CT -            #CACCAAAG   2280                                                                 - - TGCACAAGCA GTTGGTACGT GATAATGCAG ATCTTCGCTG TGAGCTTCCT AA -            #GTTAGAGA   2340                                                                 - - AACGGCTTAG AGCTACTGCA GAAAGAGTGA AAGCTTTGGA GTCAGCCCG  - #                 2389                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - AGTGGCTGGA AAACGAGCTA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CTTGATCCCT TCTGGTTGAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - AGTGGCTTGA AAATGAGCTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CTTGATCCCT TCTGGTAGAT G           - #                  - #                      - #21                                                                 __________________________________________________________________________

We claim:
 1. An antibody raised against a synthetic peptide having anamino acid sequence corresponding to from about 6 amino acids to all ofthe amino acid sequence encoded by a genetic suppressor element (GSE)produced according to a method for identifying genetic suppressorelements that confer a selectable phenotype upon a eukaryotic cell,wherein the method comprises the steps of:(a) synthesizing randomlyfragmented cDNA prepared from the total mRNA of a cell to yield DNAfragments; (b) transferring the DNA fragments to an expression vector toyield a genetic suppressor element library, wherein each of the DNAfragments is operatively linked to a protein translation initiationcodon, and wherein the expression vector expresses the DNA fragments ina living eukaryotic cell that is capable of exhibiting the selectablephenotype; (c) genetically modifying living cells by introducing thegenetic suppressor element library into the living eukaryotic cells; (d)isolating or enriching for genetically modified living eukaryotic cellscontaining genetic suppressor elements that confer the selectablephenotype by selecting cells that express the selectable phenotype; (e)obtaining the genetic suppressor element from the genetically modifiedcells,wherein said antibody is specific for an epitope comprising agenetic suppressor element that is not antigenic in the wildtypecellular protein from which the GSE is derived.
 2. An antibody accordingto claim 1 wherein the GSE corresponding to the synthetic peptide is asense-oriented GSE encoding a peptide.
 3. An antibody raised against asynthetic peptide having an amino acid sequence corresponding to fromabout 6 amino acids to all of the amino acid sequence encoded by agenetic suppressor element (GSE) produced according to a method foridentifying genetic suppressor elements corresponding to genes that whensuppressed by GSEs, confer a selectable phenotype upon a eukaryoticcell, wherein the method comprises the steps of:(a) obtaining genomicDNA or a total mRNA population from the cells; (b) randomly fragmentingthe genomic DNA or synthesizing randomly fragmented cDNA from the totalmRNA to produce a population of randomly fragmented DNA fragments; (c)ligating the randomly fragmented DNA fragments to synthetic adaptors toproduce amplifiable random DNA fragments; (d) amplifying the amplifiablerandom DNA fragments to provide a mixture of amplified DNA fragments;(e) cloning the mixture of amplified DNA fragments into a suitableexpression vector to produce a random fragment expression library; (f)transferring the random fragment expression library into appropriatetarget cells; (g) isolating or enriching for genetically modified livingcells containing a selectable phenotype-conferring genetic suppressorelement by selecting or enriching for cells that express the selectablephenotype; and (h) recovering the GSE from the target cell having theselectable phenotype,wherein said antibody is specific for an epitopecomprising a genetic suppressor element that is not antigenic in thewildtype cellular protein from which the GSE is derived.
 4. An antibodyaccording to claim 3 wherein the GSE corresponding to the syntheticpeptide is a sense-oriented GSE encoding a peptide.
 5. An antibodyaccording to claim 1 wherein the GSE confers the selectable phenotype ofresistance in a eukaryotic cell to one or more chemotherapeutic drugs.6. An antibody according to claim 1 wherein the synthetic peptide havingan amino acid sequence comprising from about 6 to all of the amino acidsencoded by a GSE identified by Seq. ID Nos. 2, 3, 4, 5, 9, 10, 12, 13,16 or
 17. 7. A synthetic peptide having an amino acid sequencecomprising from about 6 to all of the amino acids encoded by a GSEidentified by Seq. ID Nos. 2, 3, 4, 5, 9, 10, 12, 13, 16 or 17.